PROJECT SUMMARY How fetal ?-globin is turned off in adult erythroid cells is a central question of the globin switch. A clearer understanding of the underlying mechanisms forms the basis for clinical reactivation of fetal hemoglobin (HbF) - a therapeutic approach to treat ?-hemoglobin disorders such as sickle cell disease and ?-thalassemia, two of the most prevalent genetic diseases worldwide. Previous studies have established the role of BCL11A as a major repressor of ?-globin, but the mechanism remains elusive. This research focuses on mechanistic dissection of how BCL11A turns off ?-globin expression, and the identification of novel factors involved in the globin switch. My broad and long-term objectives are to enhance our basic understanding of developmental gene control, and thereby accelerate drug discovery to benefit public health. During the mentored phase, the detailed mechanism of BCL11A action will be studied using powerful newly developed technologies. Aim 1 asks how BCL11A competes with transcriptional activators at the ?-globin promoters. Use of the SMASh-BCL11A degradation system enables reversible control of BCL11A degradation, as opposed to gene knockouts where one can only examine effects of loss after a long intervening time period. CUT&RUN will be employed to map BCL11A and transcriptional activator binding at high resolution during controlled BCL11A degradation and re-expression. Aim2 examines the role of BCL11A in organizing chromatin interactions at the ?-globin locus using CAPTURE-3C-seq, which maps chromatin interactions at high resolution and sensitivity. Aim 3 will explore the functional roles of the phase separation phenomenon of the Intrinsically Disordered Regions (IDRs) of BCL11A. Phase separation is a recent discovery that represents a paradigm changing view of how biomolecules carry out their function in vivo. My study will focus on whether BCL11A phase separation regulates globin gene expression. These studies will provide mechanistic insights of how BCL11A facilitates globin switching, which is the foundation of therapeutic targeting of BCL11A to treat diseases. My long-term career goal is to become a productive and impactful scientist in the field of epigenetic and transcriptional gene control during development and in hematological diseases. To achieve this goal, I have developed a detailed scientific and career development plan for the mentored phase of the award, which is designed to enhance my skills as both investigator and group leader. I have invited two established and one junior investigator to serve on my advisory committee and guide my training. Proposed activities include courses, meetings and practices, and mostly take place at Boston Children's Hospital and surrounding institutions, including Harvard Medical School and Dana-Farber Cancer Institute. The superb training environment provided by these institutions, the invaluable critique and support from my mentor and advisory committee, and the support from this K99 award will greatly advance development of those professional skills necessary to make a successful transition to an independent investigator.